We previously showed that the NS3 protease domain is required for NS2A/NS2B and NS2B/NS3 cleavage. In addition, we shoed that NS2B is also required for NS2A/NS2B, NS2B/NS3 and NS3/NS4A cleavage, and that NS2B can function in trans. DEN4 NS2B contains 130 amino acids, most of which are hydrophobic. We have proposed that NS2B plus NS3 interact to form a two component protease. Using polymerase chain reaction (PCR), we introduced seven new unique restriction enzyme sites into NS2B (5'-Nhe I, Stu I, Afl II, Apa I, Nru I, Spe I, Bst EII-3'). These new sites did not change the amino acid sequence of NS2B. In addition to the seven unique sites introduced, there is a unique Bgl II site in NS2B, located between the Apa I and Nru I sites. Oligonucleotides were used to bridge between the nearest-neighbor pairs of all 8 sites, creating seven internal deletion mutants of NS2B. Additional recombinants designed to further define the critical residues of NS2B have also been constructed. Analysis of this series of deletion mutants showed that a 40 amino acid domain is sufficient to mediate cleavage of the resulting NS2B-NS3 polyprotein. The critical NS2B sequence contains several charged amino acid residues, presumably important for interactions with the NS3 protease components.